Rocky Mountain spotted fever became reportable in the United States nearly a century ago and cases are currently at their highest levels. However, it is not widely recognized that rickettsiae other than Rickettsia rickettsii can cause spotted fevr in the United States. Although other spotted fever group rickettsiae (SFGR) have been identified over the last two decades, there remains a gap in existing knowledge of the epidemiology of spotted fever rickettsiosis, nomenclature CDC now uses in place of Rocky Mountain spotted fever. Rickettsia parkeri, which is transmitted by Amblyomma maculatum, recently emerged as a contributor to spotted fever rickettsiosis. However, Candidatus Rickettsia andeanae, a novel SFGR also present in A. maculatum, is poorly understood and its disease implications unknown. How interactions between sympatric rickettsiae within the same tick species may affect rickettsial transmission and maintenance, has not been explored in the A. maculatum-SFGR system. In fact, this tick-rickettsial system may differ from those previously described for Dermacentor ticks and SFGR. The long-term goal of this research is to understand the biology and relationships between SFGR in ticks, using the A. maculatum-SFGR system as a model, in order to more accurately portray the complex epidemiology of spotted fever rickettsiosis. To move toward this goal, the overall objective of this R15 AREA application is to evaluate the potential contribution of R. parkeri and Ca. R. andeanae in A. maculatum to spotted fever rickettsiosis by understanding interactions between these SFGR in A. maculatum during tick feeding on vertebrates. The central hypothesis is that Ca. R. andeanae will not be infectious but will affect transmission and maintenance of R. parkeri. The rationale for the proposed research is that understanding how SFGR other than R. rickettsii contribute to disease, and to transmission and maintenance of rickettsiae, will help clarify spotted fever rickettsiosis epidemiology. The A. maculatum-SFGR system provides an ideal model with which to evaluate tick-rickettsia interactions. In keeping with AREA goals, students will participate in the proposed research on emerging vector-borne organisms without being exposed to highly pathogenic species. In this revised R15 application, two Aims are proposed to accomplish the goals: (1) to evaluate tissue tropism of Ca. R. andeanae and R. parkeri in singly and co-infected A. maculatum, and effect(s) of Ca. R. andeanae on transmission of R. parkeri; and (2) to evaluate co-feeding and Ca. R. andeanae presence in enabling tick acquisition of R. parkeri. The proposed research is significant because it promotes further understanding of potentially novel tick-rickettsia interactions. This knowledge will assist in accurately evaluating spotted fever rickettsiosis epidemiologic data. Ultimately, rational interpretation of these data will be useful in public health policy making. Finally, the proposed research is innovative because preliminary data challenge the presumption that transovarial interference occurs in all tick-borne SFGR, suggesting that co-infections may prevail in some systems. Thus, microbial interactions in arthropods may be more complex than is currently believed.